Process for the production of alpha-human atrial natriuretic polypeptide

ABSTRACT

The present invention relates to a process for the production of α-human atrial natriuretic polypeptide by recombinant DNA technology.

[0001] This invention relates to a new process for the production ofα-human atrial natriuretic polypeptide (hereinafter referred to as theabbreviation “α-hANP”) by recombinant DNA technology. More particularly,it relates to a new process for the production of α-hANP by recombinantDNA technology, to chemically synthesized genes for α-hANP andprotective peptide-fused α-hANP and to a corresponding recombinantvector and transformant comprising the same.

[0002] The α-hANP is a known polypeptide having a diuretic, natriuretic,vasorelaxant and antihypertensive activities. Therefore, it may beuseful in clinical treatment of hypertension as antihypertensivediuretic agent and has the following structure:

[0003] (Cf. Biochemical and Biophysical Research Communications Vol.118,page 131 (1984)).

[0004] The inventors of this invention have newly created a process forthe production of α-hANP by recombinant DNA technique using anexpression vector comprising a synthetic gene encoding the amino acidsequence (I) of α-hANP. According to this process, α-hANP can beobtained in high yield.

[0005] This invention provide a process for the production of α-hANP by(1) culturing a microorganism transformed with an expression vectorcomprising a synthetic gene encoding an amino acid sequence of aprotective peptide-fused α-hANP in a nutrient medium, (2) recovering theprotective peptide-fused α-hANP from the cultured broth and (3) removingthe protective peptide part of the protective peptide-fused α-hANP.

[0006] In the above process, particulars of which are explained in moredetail as follows.

[0007] The microorganism is a host cell and may include bacteria, fungi,cultured human and animal cells and cultured plant cells. Preferredexamples of the microorganism may include bacteria especially a strainbelonging to the genus Escherichia (e.g. E. coli HB101 (ATCC 33694), E.coli 294 (ATCC 31446), E. coli χ 1776 (ATCC 31537), etc).

[0008] The expression vector is usually composed of DNA having at leasta promoter-operater region, initiation codon, synthetic protectivepeptide gene, synthetic α-hANP gene, termination codon(s) andreplicatable unit.

[0009] The promoter-operater region comprises promoter, operater andShine-Dalgarno (SD) sequence (e.g. AAGG, etc.). The distance between SDsequence and intiation codon is preferably 8-12 b. p. and in the mostpreferable case as shown in the working Examples mentioned below, thedistance between SD sequence and initiation codon (ATG) is 11 b.p.Examples of the promoter-operater region may include conventionallyemployed promoter-operater region (e.g. lactose-operon, PL-promoter,trp-promoter, etc.) as well as synthetic promoter-operater region.Preferred examples of the promoter-operater region are synthetic trppromoter I, II and III which were newly synthesized by the inventors ofthis invention and DNA sequences thereof are shown in FIGS. 1, 2 and 3,respectively. In the process, there may be used 1-3 consecutivepromoter-operater region(s) per expression vector.

[0010] Preferred initiation codon may include methionine codon (ATG).

[0011] The protective peptide gene may include DNA sequencecorresponding to any of peptide or protein which is capable of forming afused protein with α-hANP and inhibiting undesired degradation of thefused protein in the host cell or the cultured broth. One of preferredexamples is “peptide Cd gene” linked to “LH protein gene” (hereinafter“the peptide Cd gene linked to LH protein gene” is referred to as“peptide CLa gene”), DNA sequence of which is shown in FIG. 4.

[0012] The DNA sequence of α-hANP gene is designed from the amino acidsequence of α-hANP, subjected to a number of specific non-obviouscriteria. Preferred example of DNA sequence of α-hANP gene is shown inFIG. 5. In the working Examples as mentioned below, between the α-hANPgene and the protective peptide gene, a DNA sequence encoding amino acidlysine is inserted, with the purpose of Achromobacter protease Idigestion at the junction of the fused protein.

[0013] The termination codon(s) may include conventionally employedtermination codon (e.g. TAG, TGA, etc.).

[0014] The replicatable unit is a DNA sequence capable of replicatingthe whole DNA sequence belonging thereto in the host cells and mayinclude natural plasmid, artificially modified plasmid (e.g. DNAfragment prepared from natural plasmid) and synthetic plasmid andpreferred examples of the plasmid may include plasmid pBR 322 orartificially modified thereof (DNA fragment obtained from a suitablerestriction enzyme treatment of pBR 322). The replicatable unit maycontain natural or synthetic terminator (e.g. synthetic fd phageterminator, etc.).

[0015] Synthetic preparation of promoter-operater region, initiationcodon, protective peptide gene, α-hANP gene and termination codon can beprepared in a conventional manner as generally employed for thepreparation of polynucleotides.

[0016] The promoter-operater region, initiation codon, protectivepeptide gene, α-hANP gene and termination codon(s) can consecutively andcircularly be linked with an adequate replicatable unit (plasmid)together, if desired using an adequate DNA fragment(s) (e.g. linker,other restriction site, etc.) in a conventional manner (e.g. digestionwith restriction enzyme, phosphorylation using T4 polynucleotide kinase,ligation using T4 DNA-ligase) to give an expression vector.

[0017] The expression vector can be inserted into a microorganism (hostcell). The insertion can be carried out in a conventional manner (e.g.transformation, microinjection, etc.) to give a transformant.

[0018] For the production of α-hANP in the process of this invention,thus obtained transformant comprising the expression vector is culturedin a nutrient medium.

[0019] The nutrient medium contains carbon source(s) (e.g. glucose,glycerine, mannitol, fructose, lactose, etc.) and inorganic or organicnitrogen source(s) (ammonium sulfate, ammonium chloride, hydrolysate ofcasein, yeast extract, polypeptone, bactotrypton, beef extracts, etc.).If desired, other nutritious sources (e.g. inorganic salts (e.g. sodiumor potassium biphosphate, dipotassium hydrogen phosphate, magnesiumchloride, magnesium sulfate, calcium chloride), vitamins (e.g. vitaminB1), antibiotics (e.g. ampicillin), etc.) may be added to the medium.

[0020] The culture of transformant may generally be carried out at pH5.5-8.5 (preferably pH 7-7.5) and 18-40° C. (preferably 25-38° C.) for5-50 hours.

[0021] Since thus produced protective peptide-fused α-hANP generallyexists in cells of the cultured transformant, the cells are collected byfiltration or centrifuge, and cell wall and/or cell membrane thereof isdestroyed in a conventional manner (e.g. treatment with super sonicwaves and/or lysozyme, etc.) to give debris. From the debris, theprotective peptide-fused α-hANP can be purified and isolated in aconventional manner as generally employed for the purification andisolation of natural or synthetic proteins (e.g. dissolution of proteinwith an appropriate solvent (e.g. 8M aqueous urea, 6M guanidine, etc.),dialysis, gel filtration, column chromatography, high performance liquidchromatography, etc.).

[0022] The α-hANP can be prepared by cleaving the protectivepeptide-fused α-hANP in the presence of an appropriate protease (e.g.Achromobacter Protease I(AP I), etc.) treatment or chemical method (e.g.treatment with cyanogen bromide). In the case where C-terminal of theprotective peptide is lysine, there can preferably be employed treatmentwith API. Although API is a known enzyme (Cf. Biochim. Biophys. Acta.,660, 51 (1981)), it has never been reported that fused proteins preparedvia recombinant DNA technology can preferably be cleaved by thetreatment with API. This method may preferably be employed for cleavinga fused protein composed of peptides having a lysine between aprotective peptide and a target peptide having no lysine in itsmolecule.

[0023] The cleavage of the fused protein may be carried out at pH 5-10and 20-40° C. (preferably 35-40° C.) for 2-15 hours in an aqueoussolution (e.g. buffer solution, aqueous urea, etc.).

[0024] In the working Examples as mentioned below, the fused protien istreated with API firstly in a buffer solution containing 8M urea at pH5, secondly, in a buffer solution containing 4M urea at pH 9. In thiscondition, the fused protein is cleaved at lysine site, and the producedα-hANP is refolded spontaneously.

[0025] Thus produced α-hANP can be purified and isolated from theresultant reaction mixture in a conventional manner as mentioned above.

[0026] The Figures attached to this specification are explained asfollows.

[0027] In the some of Figures, oligonucleotides are illustrated with thesymbol z,1 or

(in this symbol, the mark  means 5′-phosphorylated end by T4polynucleotide kinase), and blocked oligonucleotides are illustratedwith the symbol,

or

(in this symbol, the mark Δ means ligated position).

[0028] In the DNA sequence in this specification, A, G, C and T mean theformula:

[0029] respectively, and

[0030] 5′-terminal A, G, C and T mean the formula:

[0031] respectively, and

[0032] 3-terminal A, G, C and T mean the formula;

[0033] respectively, unless otherwise indicated.

[0034] In the following Examples, following abbreviations are used.

[0035] Ap, Gp, Cp and Tp mean the formula:

[0036] respectively, and

[0037] 3′-teminal AOH, GOH, COH and TOH mean the formula:

[0038] respectively, and

[0039] 5′-terminal HOAp, HOGp, HOCp and HOTp mean the formula:

[0040] respectively, and

[0041] A^(Bz)po, G^(iB)po, C^(Bz)po, Tpo and TO mean the formula:

[0042] respectively, and

[0043] DMTR is dimethoxytrityl, and

[0044] CE is cyanoethyl.

[0045] Mono (or di, or tri)mer (of oligonucleotides) can be prepared by,for examples the Hirose's method [Cf. Tanpakushitsu Kakusan Kohso 25,255 (1980)] and coupling can be carried out, for examples on celluloseor polystyrene polymer by a phosphotriester method [Cf. Nucleic AcidResearch, 9, 1691 (1981), Nucleic Acid Research 10, 1755 (1982)].

[0046] The following Examples are given for the purpose of illustratingthis invention, but not limited thereto.

[0047] In the Examples, all of the used enzymes (e.g. restrictionenzyme, T4 polynucleotide kinase, T4 DNA ligase) are commerciallyavailable and conditions of usage of the enzymes are obvious to theperson skilled in the art, for examples, referring to a prescriptionattached to commercially sold enzymes.

[0048] Further, in the Examples, the term “polystyrene polymer” meansaminomethylated polystyrene.HCl, divinylbenzene 1%, 100-200 mesh (soldby Peptide Institute Inc.)

EXAMPLE 1

[0049] Synthesis of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH (AH7)

[0050] (1) Synthesis of DMTrOTpoC^(Bz)poTO-succinyl-polystyrene polymer

[0051] i) Preparation of HOTO-succinyl polystyrene polymer:

[0052] To a DMTrO-TO-succinyl-polystyrene polymer (51.8 mg, 10.37μ mole)(prepared by the method described in Nucleic Acid Research 10, 1755(1982)) in a reaction syringe, 5% dichloroacetic acid (DCA) solution indichloromethane (2 ml) was added. After the standing for 1 minute, themixture was filtered through filter glass by nitrogen gas. The DCAtreatment was repeated more two times. The polymer was washed withdichloromethane (2 ml×3), methanol (2 ml×3) and pyridine (2 ml×3)succesively, and dried by nitrogen gas stream to give polymer adduct I.

[0053] ii) Preparation of DMTrOTpoC^(Bz)po-:

[0054] DMTrOTpoC^(Bz)po-CE (32.4 mg, 8.12 μmole) prepared by the methoddescribed in Tanpakushitsu Kakusan Kohso 25, 255 (1980) was treated witha mixture of triethylamine and acetonitrile (1:1 v/v, 5 ml) at roomtemperature for 30 minutes. The phosphodiester dimer (DMTrOTpOC^(Bz)po-)thus obtained was dried, water being separated as the pyridine azeotrope(2 ml×2).

[0055] iii) Coupling:

[0056] The dimer (DMTrOTpoC^(Bz)po-) and mesitylensulfonylnitrothiazolide (MSNT) (80 mg) were dissolved in pyridine (0.5ml). The solution was added into the reaction syringe with the polymeradduct I, and the mixture was shaked for 1 hour at room temperature. Thereaction mixture was filtered through filter glass by nitrogen gas, andwashed with pyridine (2 ml×3) to give the polymer adduct II.

[0057] iv) Acetylation of Unreacted 5′-hydroxy Groups:

[0058] To the polymer adduct II obtained as above, pyridine (0.9 ml) andacetic anhydride (0.1 ml) were added and the mixture was shaked for 15minutes. Then the reaction solution was removed through filter glass andthe resultant polymer was washed successively with pyridine (2 ml×3),methanol (2 ml×3) and dichloromethane (2 ml×3), and then dried bynitrogen gas stream. The polymer adduct(DMTrOTpoC^(Bz)poTO-succinyl-polystyrene polymer) can use for the nextcoupling step.

[0059] (2) Synthesis ofDMTrOTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrene polymer:

[0060] DMTrOTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer was synthesized from DMTrOTpoC^(Bz)poTO-succinyl-polystyrenepolymer and DMTrOTpoC^(Bz)poC^(Bz)poCE (44.9 mg) according to similarconditions as above (1).

[0061] (3) Synthesis ofDMTrOA^(Bz)poG^(iB)poA^(Bz)poTpoC^(Bz)poC^(Bz)poTpo-C^(Bz)poTO-succinyl-polystyrenepolymer:

[0062]DMTrOA^(Bz)poG^(iB)poA^(Bz)poTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer was synthesized fromDMTrOTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrene polymer andDMTrOA^(Bz)poG^(iB)poA^(Bz)poCE (48.5 mg) according to similarconditions as above (1).

[0063] (4) Synthesis ofDMTrOC^(Bz)poG^(iB)poTpoA^(Bz)poG^(iB)poA^(Bz)poTpo-C^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer:

[0064]DMTrOC^(Bz)poG^(iB)poTpoA^(Bz)poG^(iB)poA^(Bz)poTpoC^(Bz)poC^(Bz)poTpo-C^(Bz)poTO-succinyl-polystyrenepolymer was synthesized fromDMTrOA^(Bz)poG^(iB)poA^(Bz)poTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer and DMTrOC^(Bz)poG^(iB)poTpoCE (45.1 mg) according to similarconditions as above (1).

[0065] (5) Synthesis ofDMTrOC^(Bz)poTpoG^(iB)poC^(Bz)poG^(iB)poTpoA^(Bz)po-G^(iB)poA^(Bz)poTpoC^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer:

[0066]DMTrOC^(Bz)poTpoG^(iB)poC^(Bz)poG^(iB)poTpoA^(Bz)poG^(iB)poA^(Bz)poTpo-C^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer (60 mg) was synthesized fromDMTrOC^(Bz)poG^(iB)poTpoA^(Bz)poG^(iB)poA^(Bz)poTpo-C^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer and DMTrOC^(Bz)poTpoG^(iB)poCE (45.1 mg) according to similarconditions as above (1). At this final step, unreacted 5′-hydroxy groupwas not necessary to protect with an acetyl group.

[0067] (6) Synthesis of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH:

[0068]DMTrOC^(Bz)poTpoG^(iB)poC^(Bz)poG^(iB)poTpoA^(Bz)poG^(iB)poA^(Bz)poTpo-C^(Bz)poC^(Bz)poTpoC^(Bz)poTO-succinyl-polystyrenepolymer (60 mg) was treated with 1M N,N,N′,N′-tetramethyleneguanidiumpyridine 2-aldoximate (in dioxane-water (1:1: v/v, 1 ml)) at 37° C. for20 hours in a sealed tube. To the reaction mixture 28% (w/w) aqueousammonia (12 ml) was added, and the mixture was heated at 60° C. for 5hours. The solid polymer was removed by filtration and washed with water(10 ml). The filtrate and washed solution were evaporated to dryness,and the residue was treated with 80% aqueous acetic acid (25 ml) at roomtemperature for 15 minutes. After removal of the solvent, the residuewas dissolved in 0.1M triethylammonium carbonate buffer (pH 7.5, 25 ml)was washed with diethylether (3×25 ml). Aqueous layer was evaporated todryness and the residue was dissolved in 0.1M triethylammonium carbonatebuffer (pH 7.5, 2 ml) to yield curde HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOHin the solution.

[0069] (7) Purification of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH

[0070] i) First purification of the crude product was performed bycolumn chromatography on Biogel P2 (Biolad) (24×2.6 cm ID). Thefractions corresponding to the first eluted peak (50 mM ammonium acetatecontaining 0.1 mM EDTA, flow rate: 1 ml/min) were collected andfreeze-dried to give the first purified product.

[0071] ii) Second purification of the first purified product wasperformed by high performance liquid chromatography (HPLC) on CDR-10(Mitsubishi Kasei) (25 cm×4.6 mm ID) using a linear gradient of 1Mammonium acetate-10% (v/v) aqueous ethanol to 4.5 M ammonium acetate-10%(v/v) aqueous ethanol (80 minutes, flow rate: 1 ml/minute, 60° C.) togive the second purified product.

[0072] iii) Third purification of the second purified product wasperformed by reverse phase HPLC (Rp-18-5μ(×77) (Merck), 15 cm×4 mm ID)using a linear gradient of 0.1 M ammonium acetate to 0.1 M ammoniumacetate-15% (v/v) aqueous acetonitrile (40 minutes, 1.5 ml/minute, roomtemperature) to give the final purified product.

[0073] (HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH)

[0074] (8) Analysis of oligonucleotide:

[0075] (HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH)

[0076] i) Digestion by phosphodiesterase

[0077] The mixture of HOCpTpGpCpGpTpApGpApTpCpCpTpCpTOH (10 μg, 5.1 μl),0.2M MgCl₂ (20 μl), 0.2M tris-HCl (pH8.5) (20 μl) and 0.1 mM EDTA (144.9μl) was treated with phosphodiesterase (10 unit, 10 μl) at 37° C. for 20minutes, and then heated at 100° C. for 2 minutes.

[0078] ii) Analysis by HPLC:

[0079] The oligonucleotide in the reaction mixture was analyzed by HPLC(CDR-10 (Mitsubishi Kasei), 25 cm×4.6 mm ID) using a linear gradient ofwater to 2.0 M ammonium acetate (pH 3.4) (40 minutes, flow rate: 1.5ml/minute, 60° C.). From each peak area observed, its nucleotidecomposition was determined comparing with area of a standard sample.

[0080] Calcd: pCOH 4.000, pAOH 2.000, pTOH 5.000, pGOH 3.000

[0081] Observed: pCOH 3.770, pAOH 2.026, pTOH 5.237, pGOH 2.968

EXAMPLE 2

[0082] Synthesis of oligonucleotide:

[0083] Following oligonucleotides were prepared in a similar manner tothat described in Example 1. (1) HOApGpCpTpTpGpApApGpTpTpGpApGpCpApTpGOH(AH1) (2) HOApApTpTpCpApTpGpCpTpCpApApCpTpTpCpAOH (AH2) (3)HOApApTpTpCpGpGpTpApTpGpGpGpCOH (AH3) (4)HOTpTpCpApCpCpGpCpCpCpApTpApCpCpGOH (AH4) (5)HOGpGpTpGpApApGpCpTpApApApTpCpTOH (AH5) (6)HOCpGpCpApGpApGpApTpTpTpApGpCOH (AH6) (7)HOApApGpCpApApGpApGpGpApTpCpTpAOH (AH8) (8)HOTpGpCpTpTpTpGpGpTpGpGpCpCpGpTOH (AH9) (9)HOTpCpCpApTpApCpGpGpCpCpApCpCpAOH (AH10) (10)HOApTpGpGpApCpCpGpCpApTpCpGpCpTOH (AH11) (11)HOTpGpApGpCpApCpCpGpApTpGpCpGpGOH (AH12) (12)HOGpCpTpCpApGpTpCpCpGpGpTpCpTpGOH (AH13) (13)HOCpApGpCpCpCpApGpApCpCpGpGpApCOH (AH14) (14)HOGpGpCpTpGpTpApApCpTpCpTpTpTpCOH (AH15) (15)HOTpApApCpGpGpApApApGpApGpTpTpAOH (AH16) (16)HOCpGpTpTpApCpTpGpApTpApGOH (AH17) (17) HOGpApTpCpCpTpApTpCpApGOH (AH18)

EXAMPLE 3

[0084] Synthesis of oligonucleotides:

[0085] Following oligonucleotides were prepared by a similar manner tothat of Example 1. (1) HOApApTpTpTpGpCpCpGpApCpAOH (A) (2)HOCpGpTpTpApTpGpApTpGpTpCpGpGpCpAOH (B) (3)HOTpCpApTpApApCpGpGpTpTpCpTpGpGpCOH (C) (4)HOGpApApTpApTpTpTpGpCpCpApGpApApCOH (D) (5)HOApApApTpApTpTpCpTpGpApApApTpGpAOH (E) (6)HOTpCpApApCpApGpCpTpCpApTpTpTpCpAOH (F) (7)HOGpCpTpGpTpTpGpApCpApApTpTpApApTOH (G) (8)HOGpTpTpCpGpApTpGpApTpTpApApTpTpGOH (H) (9)HOCpApTpCpGpApApCpTpApGpTpTpApApCOH (I) (10)HOGpCpGpTpApCpTpApGpTpTpApApCpTpAOH (J) (11)HOTpApGpTpApCpGpCpApApGpTpTpCpApCOH (K) (12)HOCpTpTpTpTpTpApCpGpTpGpApApCpTpTOH (L) (13)HOGpTpApApApApApGpGpGpTpApTOH (M′) (14) HOCpGpApTpApCpCOH (N′) (15)HOGpTpApApApApApGpGpGpTpApTpCpGOH (M) (16) HOApApTpTpCpGpApTpApCpCOH (N)(17) HOApApTpTpCpApTpGpGpCpTOH (SA) (18)HOGpGpTpTpGpTpApApGpApApCpTpTpCpTOH (SB) (19)HOTpTpTpGpGpApApGpApCpTpTpTOH (SC) (20)HOCpApCpTpTpCpGpTpGpTpTpGpApTpApGOH (SD) (21)HOTpTpApCpApApCpCpApGpCpCpApTpGOH (SE) (22)HOCpCpApApApApGpApApGpTpTpCOH (SF) (23)HOCpGpApApGpTpGpApApApGpTpCpTpTOH (SG) (24)HOGpApTpCpCpTpApTpCpApApCpAOH (SH)

EXAMPLE 4

[0086] Synthesis of oligonucleotides:

[0087] Following oligonucleotides were prepared by a similar manner tothat of Example 1. (1) HOApApCpTpApGpTpApCpGpCOH (Np1) (2)HOApApCpTpTpGpCpGpTpApCpTpApGpTpTOH (Np4) (3)HOApApGpTpTpCpApCpGpTpApApApApApGOH (Np2) (4)HOApTpApCpCpCpTpTpTpTpTpApCpGpTpGOH (Np5) (5)HOGpGpTpApTpCpGpApTpApApApApTpGOH (Np3) (6)HOGpTpApGpApApCpApTpTpTpTpApTpCpGOH (Np6) (7)HOTpTpCpTpApCpTpTpCpApApCpApApAOH (Cd1) (8)HOGpGpTpCpGpGpTpTpTpGpTpTpGpApAOH (Cd2) (9)HOCpCpGpApCpCpGpGpCpTpApTpGOH (Cd3) (10)HOGpCpTpGpGpApGpCpCpApTpApGpCpCOH (G2) (11)HOGpCpTpCpCpApGpCpTpCpTpCpGpTpCOH (H1) (12)HOCpGpGpTpGpCpGpCpGpApCpGpApGpAOH (H2) (13)HOGpCpGpCpApCpCpGpCpApGpApCpTpGOH (I1) (14) HOGpApTpApCpCpApGpTpCpTpGOH(Cd4) (15) HOGpTpApTpCpGpTpApGpApCpGOH (Cd5) (16)HOApCpCpCpTpCpGpTpCpTpApCOH (Cd6) (17) HOApGpGpGpTpGpGpCpGpApTpGOH (Cd7)(18) HOApApTpTpCpApTpCpGpCpCOH (Cd8)

EXAMPLE 5

[0088] Construction and Cloning of the Synthetic trp promoter II Gene(as Illustrated in FIGS. 6 and 7):

[0089] The trp promoter II gene was constructed by the similar method asdescribed in Example 7 (as illustrated in FIG. 6). The synthetic genewas ligated with EcoRI-BamHI fragment of pBR322 (commercially available:Takarashuzo, NEB, etc.) and then E. coli HB101 (ATCC 33694) wastransformed with the ligation product. The plasmid obtained from thetransformant of ^(R)Amp and ^(S)Tet was digested with HpaI to confirm aband (4.1 kbp), and then digested with BamHI to confirm a band of 90b.p. on PAGE. Moreover, the fragment of 56 b.p. by EcoRI-BamHI digestionwas confirmed by the comparison with size marker on PAGE. This plasmidwas named pTrpEB7 and used construction of expression vector.

EXAMPLE 6

[0090] Construction and Cloning of trp promoter vector (pBR322trp) (asIllustrated in FIG. 8):

[0091] Plasmid pBR322 (9 μg) was digested with EcoRI and BamHIrestriction endnucleases. Reaction was terminated by heating at 65° C.for 5 minutes and the fragments were separated by electrophoresis on a0.8% agarose gel to give the small fragment (500 ng) of 375 b.p. On theother hand, plasmid pTrpEB7 (10 μg) was digested with EcoRI and BamHI,followed by preparative gel electrophoresis to give the large fragment(5 μg) of 4094 b.p. The pTrpEB7 EcoRI-BamHI fragment (4094 b.p., 200 μg)was ligated with the pBR322 EcoRI-BamHI fragment (375 b.p., 100 ng) inthe ligation buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 20 mM DTT, 1mM ATP, 1 mM spermidine, 50 μg/ml BSA) (20 μl) containing T4 DNA ligase.(Takarashuzo: 360 unit) at 15° C. overnight. The ligated mixture wastransformed into E. coli HB101 by Kushiner's method (Cf. T. Maniatis etal Molecular Cloning p252 (1982), Cold Spring Harbor Laboratory) andtetracycline resistant transformants were obtained on the platecontaining tetracycline (25 μg/ml). The plasmid pBR322trp isolated fromthe transformant was digested with EcoRI-BamHI (375 b.p., 4094 b.p.) andHpaI (4469 b.p.) to confirm the trp promoter gene by 7.5% PAGE and 0.8%agarose gel electrophoresis.

EXAMPLE 7

[0092] Construction of the Synthetic trp promoter III Gene (asIllustrated in FIG. 9):

[0093] Each oligonucleotides (B-M′) (each 0.2 n mole) of block I′, II′and III′ were phosphorylated with T4 polynucleotide kinase (BRL; 2.5unit) in the ligation buffer (70 μl) at 37° C. for 1 hour. To thereaction mixture of each blocks T4 DNA ligase (300 unit) and 20 mM ATP(2 μl) were added, and the mixture was incubated at 15° C. for 30minutes. The reaction was terminated by heating at 65° C. for 10minutes. The reaction mixture of these blocks (I′, II′ and III′) was puttogether and mixed with unphosphorylated oligonucleoties (A, N′) in thepresence of T4 DNA ligase (360 unit) and 20 mM ATP (2 μl). After theincubation of the mixture at 15° C. for 1 hour, the last ligationproduct was purified by 2-16% gradient polyacrylamide gelelectrophoresis (PAGE) to give the 106 b.p. synthetic trp promoter IIIgene.

EXAMPLE 8

[0094] Construction and Cloning of Double trp promoter vector(p322dtrpS)(as Illustrated in FIG. 10):

[0095] Plasmid pBR322trp was digested with EcoRI and ClaI, followed bypreparative agarose gel electrophoresis to give the large fragment of4446 b.p. This fragment (4446 b.p.) was ligated with trp promoter IIIgene (106 b.p.) obtained in Example 7 in the presence of T4 DNA ligase.The ligated mixture was transformed into E. coli HB101 to give thetransformants of ampicillin and tetracycline resistance. The plasmidp322dtrpS obtained from the transformant was confirmed by restrictionendonuclease analysis ClaI-BamHI (352 b.p.), HpaI (107 b.p.) andAatII-ClaI (287 b.p.).

EXAMPLE 9

[0096] Construction of Peptide Cd Gene With a Part of DNA Fragment ofSynthetic trp promoter III (as Illustrated in FIGS. 11 and 12):

[0097] Each oligonucleotides (0.2 n mole) (Np1-Cd8, shown in Example 4)of block I″, II″ and III″ were phosphorylated with T4 polynucleotidekinase (2.5 unit) in ligation buffer (60 μl) at 37° C. for 1 hour. Tothe reaction mixture of each block T4 DNA ligase (360 unit) and ATP (2μl) was added, the mixture was incubated at 15° C. for 1 hour. Thereaction mixture of these blocks (I″, II″ and III″) was put together andincubated with T4 DNA ligase (360 unit) and 20 mM ATP (2 μl) at 15° C.overnight, and then heated at 80° C. for 10 minutes. To the mixture 500mM NaCl (20 μl) and EcoRI (20 unit) were added. After the incubation at37° C. for 2 hours, the last ligation product was purified by 15% PAGEto give the peptide Cd gene with a part of DNA fragment of synthetic trppromoter III (125 b.p.), DNA sequence of which is illustrated in FIG.12.

EXAMPLE 10

[0098] Construction and Cloning of plasmid pCdγ (as Illustrated in FIG.13):

[0099] Plasmid pγtrp(4544 b.p.) (Cf. GB2164650A published on Mar. 26,1986; Escherichia coli F-9 containing this plasmid pγtrp has beendepositing with. FRI(Japan) under the number FERM BP-905 from Sep. 20,1984) was digested with HpaI and EcoRI to give a large fragment (4510b.p.), which was ligated with the peptide Cd gene with a part of DNAfragment of synthetic trp promoter III (125 b.p.) as obtained in Example9 in the presence of T4 DNA ligase. The ligated mixture was transformedinto E. coli HB101. The plasmid (pCdγ) obtained from the transformant of^(R)Amp was confirmed by restriction endonuclease analysis:

[0100] ClaI-BamHI (543 b.p.), ClaI-HindIII (273 b.p.), ClaI-EcoRI (93b.p.) and AatII-ClaI (180 b.p.).

EXAMPLE 11

[0101] Construction and Cloning of plasmid pCdγtrpSd (as Illustrated inFIG. 14):

[0102] The plasmid pCdγ was digested with ClaI and BamHI to give thesmaller fragment (543 b.p.), which was ligated with the ClaI-BamHIfragment (4223 b.p.) of p322dtrpS (Example 7) in the presence of T4 DNAligase. The ligated mixture was transformed into E. coli HB101. Theplasmid (pCdγtrpSd) obtained from the transformant of ^(R)Amp wasconfirmed by restriction endonuclease analysis:

[0103] HpaI-BamHI (107,575 b.p.), ClaI-BamHI (543 b.p.),

[0104] PstI-EcoRI (1057 b.p.), EcoRI-BamHI (450 b.p.)

[0105] HindIII-BamHI (270 b.p.), ClaI-HindIII (273 b.p.)

EXAMPLE 12

[0106] Preparation of α-hANP Gene With Linker DNA (as Illustrated inFIG. 15):

[0107] Each oligonucleotides (AH2-AH17) (each 0.2 n mole) of block I andII were phosphorylated with T4 polynucleotide kinase (2.5 unit) in theligation buffer (70 μl) at 37° C. for 1 hour. To the reaction mixture ofeach blocks T4 DNA ligase (300 unit) and 20 mM ATP (2 μl) were added,and the mixture was incubated at 15° C. for 30 minutes. The reaction wasterminated by heating at 65° C. for 10 minutes. The reaction mixture oftwo blocks (I′″ and II′″) was put together and mixed withunphosphorylated oligonucleotides (AH1, AH18) in the presence of T4 DNAligase (300 unit) and 20 mM ATP (2 μl). After the incubation of themixture at 15° C. for 1 hour, the last ligation product was purified by2-16% gradient PAGE to give the 134 b.p. α-hANP gene with linker DNA (asillustrated in FIG. 5).

EXAMPLE 13

[0108] Construction and Cloning of α-hANP Expression Vector pCLaHtrpSd(as Illustrated in FIG. 16):

[0109] The plasmid pCdγtrpSd was digested with HindIII and BamHI to givethe larger fragment (4743 b.p.), which was ligated with the α-hANP genewith linker DNA (134 b.p.) in the presence of T4 DNA ligase. The ligatedmixture was transformed into E. coli HB101 to give a transformant H1.The plasmid (pCLaHtrpSd) (which contains CLaH protein(peptide CLa-fusedα-hANP protein)gene, DNA sequence of which is illustrated in FIG. 17)obtained from the transformant of ^(R)Amp (E. coli H1) was confirmed byrestriction endonuclease analysis:

[0110] AatII-ClaI (287 b.p.), ClaI-BamHI (407 b.p.),

[0111] ClaI-EcoRI (93, 198 b.p.), EcoRI-BamHI (116, 198 b.p.),HindIII-BamHI (134 b.p.), HpaI-BamHI (107, 439 b.p.).

EXAMPLE 14

[0112] Expression of a Gene Coding for the peptide CLa-Fused α-hANP(CLaH Protein):

[0113] An overnight culture of E. coli H1 containing the expressionvector, plasmid pCLaHtrpSd in L broth (20 ml) containing 50 μg/mlampicillin was diluted in M9 medium (400 ml) containing 0.2% glucose,0.5% casamino acid (acid-hydrolyzed casein), 50 μg/ml vitamin B1 and 25μl/ml ampicillin, and the E. coli was cultured at 37° C. When A600(absorbance at 600 nm) of the cultured broth was 0.5, β-indole acrylicacid (2 mg/ml ethanol; 2 ml) was added and the cells were incubated for3 hours (final A600=1.85). Then the cells were harvested bycentrifugation (6000 rpm, 4° C., 5 minutes).

EXAMPLE 15

[0114] Isolation and Purification of α-hANP:

[0115] (1) Isolation and Purification of the peptide CLa-Fused α-hANP(CLaH Protein)

[0116] The wet cell paste from the cultured broth (600 ml) as preparedin Example 14 was suspended in 8 ml of 10 mM PBS-EDTA (pH 7.4) (NaCl(8.0 g), KCl (0.2 g), Na₂HPO4 12H₂O (2.9 g), KH₂PO₄ (0.2 g), EDTA (3.73g)/liter) and cells were destroyed by sonication at 0° C. The pellet wascollected by centrifugation at 15,000 rpm for 20 minutes (4° C.), andsuspended in 8 ml of 6M guanidine-HCl, 10 mM PBS-EDTA and 2 mMβ-mercaptoethanol and the suspension was treated by super sonication at0° C. The suspension was centrifuged at 15,000 rpm for 20 minutes (4°C.) and the supernatant was dialyzed overnight at 4° C. against 10 mMpBS-EDTA solution containing p-nitrophenyl methylsulfonyl fluoride(PMSF). After the fraction dialyzed was centrifuged (15,000 rpm, 4° C.,20 minites), the pellet was dissolved in 100 mM Tris-HCl buffer (pH 8.0)(8 ml) containing 6M guanidine-HCl, 10 mM EDTA and 100 mM dithiothreitoland the solution was stood overnight. The solution was dialyzed against1M acetic acid (0.5 liters) containing 10 mM 2-mercaptoethanol twice andadjusted to pH 8.0 with trisaminomethane. The resulting precipitate(fused protein; 15.2 mg) was collected by centrifugation (3.000 rpm, 10minutes), and washed with 10 mM sodium acetate buffer (pH 5.0).

[0117] (2) Elimination of peptide CLa from the peptide CLa Fused α-hANPwith Achromobacter protease I(API):

[0118] The fused protein obtained above was suspended in 10 mM sodiumacetate buffer (pH 5.0) (30 ml) containing 8M urea, the suspension wasincubated with Achromobactor protease I(API) (0.25 unit) (Wako purechemical industries, Ltd) at 37° C. for 2 hours. The reaction mixturewas diluted with distilled water (30 ml), adjusted to pH 9.0 withtrisaminomethane, and then incubated with additional API (0.25 unit) at37° C. for 2 hours. The reaction solution was diluted with 10 mM sodiumphosphate buffer (pH 7.0) (120 ml), and adjusted to pH 7 with aceticacid. The solution was applied to a Sp-sephadex C-25 column (15 ml)equilibrated with 10 mM solium phosphate buffer (pH 7.0). The column waswashed with the same buffer, and eluted with 10 mM sodium phosphatebuffer (pH 8.0) containing 0.5M aqueous sodium chloride to collect thefractions containing a partial purified α-hANP (0.4 mg).

[0119] (3) High Performance Liquid Chromatography (HPLC):

[0120] The pooled fraction obtained in the above (2) was concentrated invacuo, dialyzed against water (300 ml), and purified by reverse phaseHPLC to give a pure α-hANP (0.3 mg). HPLC condition (preparation)column: Beckman Ultrapore semi-prep. (φ10 × 250 mm) flow rate: 2.5ml/minute elution: linear gradient from 10% to 60% acetonitrile in 0.01M trifluoroacetic acid over 50 minutes. monitor absorbance at 214 nm(analysis) column: Beckmann Ultrapore RPSC (φ 4.6 × 75 mm) flow rate: 1ml/minute elution: same condition as the preparation retention time:11.9 minutes

[0121] The α-hANP was supperimposed with authentic α-hANP (sold byFunakoshi)

[0122] (4) Amino Acid Analysis of α-hANP

[0123] The sample was reduced and carboxymethylated, and then hydrolyzedwith 6N HCl at 110° C. for 24 hours. The amino acid composition ofα-hANP was obtained using a Waters amino acid analysis system.

[0124] Amino acid compositions (residues per mole) of α-hANP werecoincided with the expected values.

[0125] (5) Amino Acid Sequence Analysis of α-hANP

[0126] The N-terminal amino acid sequence of α-hANP was determined byEdman's method (DABITC method) [described in FEBS Lett., 93,205 (1978)]to confirm N-terminal Ser and Leu sequence. C-terminal amino acids(Ser-Phe-Arg-Tyr) were determined by the digestion with carboxypeptidaseand the followed amino acid analysis using a Waters amino acid analysissystem. The whole amino acid sequence of α-hANP obtain in the aboveExample was determined by using both procedures and was identical withthe known sequence of α-hANP.

EXAMPLE 16

[0127] Construction and Cloning of plasmid pBR322trpSs (as Illustratedin FIG. 18):

[0128] Plasmid pBR322 was digested with EcoRI and ClaI. The largefragment (4340 bp) was purified by 0.8% agarose gel electrophoresis, andligated to the synthetic trp promoter III gene in the presence of T4 DNAligase and 1 mM ATP. The ligation mixture was used to transform E. coliHB101. The plasmid DNA (pBR322trpSs) was isolated from a transformedclone ^(R)Amp) and charactarized by restriction endonuclease analysis.

[0129] Analysis data: Hpa I; 4445 bp, ClaI-Pst I; 834 bp

EXAMPLE 17

[0130] Construction and Cloning of plasmid pCLaHtrp-2 (as Illustrated inFIG. 19):

[0131] Plasmid pCLaHtrpSd was digested with ClaI and BamHI. The smallfragment (407 bp) was isolated. On the other hand pBR322trpSs wasdigested with ClaI and BamHI. The larger fragment (4093 bp) was isolatedand ligated to the former DNA (407 bp). After transformation of E. coliHB101 with the ligation mixture, the desired plasmid (pCLaHtrp-2) wasisolated from a transformed clone(^(R)Amp) and characterized byrestriction enzyme analysis: ClaI-Pst I; 834 bp, ClaI-BamHI; 407 bp

EXAMPLE 18

[0132] Synthesis of oligonucleotides:

[0133] Following oligonucleotides were prepared in a similar manner tothat of Example 1. (1) HOGpApTpCpCpTpCpGpApGpApTpCpApAOH (T1) (2)HOGpCpCpTpTpTpApApTpTpGpApTpCpTpCpGpApGOH (T2) (3)HOTpTpApApApGpGpCpTpCpCpTpTpTpTpGpGpAOH (T3) (4)HOApApApApApGpGpCpTpCpCpApApApApGpGpAOH (T4) (5)HOGpCpCpTpTpTpTpTpTpTpTpTpTpGOH (T5) (6) HOTpCpGpApCpApApApApAOH (T6)

EXAMPLE 19

[0134] Construction and Cloning of Synthetic fd phage Terminator (asIllustrated in FIGS. 20 and 21):

[0135] The synthetic fd phage terminator was constructed by a similarmethod as described in Example 7 (as illustrated in FIG. 20).

[0136] Namely, DNA oligomers T2, T3, T4 and T5 (each 0.4 nmole) weremixed and phosphorylated with T4 polynucleotide kinase in the presenceof 1 mM ATP. The reaction mixture was heated at 65° C. for 10 minutes toinactivate the enzyme. To the resultant mixture, DNA oligomer T1 and T6(each 0.8 nmole) and T4 DNA ligase were added. The mixture was incubatedat 15° C. for 30 minutes, and applied to 2→16% gradient polyacrylamidegel electrophoresis. The desired DNA fragment (47 bp) was recovered byelectroelution and ligated to the larger fragment of pBR322 digestedwith BamHI and Sal I (4088 bp). After transformation of E. coli HB101with the ligation mixture, the desired plasmid (pter) was isolated froma transformed clone (^(R)Amp).

[0137] Restriction enzyme analysis: BamHI-Sal I; 47 bp, Ava I; 817 bp

EXAMPLE 20

[0138] Construction and Cloning of α-hANP expression vector plasmidpCLaHtrp3t (as Illustrated in FIG. 22):

[0139] Plasmid pCLaHtrp-2 was digested with Pst I and BamHI. From thedigestion mixture, the small fragment (1241 bp) was isolated and ligatedto the large fragment of pter 21 obtained from digestion of pter 21 withPst I and BamHI (3005 bp).

[0140] The ligation mixture was transformed into E. coli HB101 to give atransformant E. coli H2. The plasmid CLaHtrp3t (which contains CLaHprotein gene) obtained from the transformant of ^(R)Amp (E. coli H2) wasconfirmed by restriction endonuclease analysis: ClaI-EcoRI; 93 bp, 198bp, HindIII-BamHI; 134 bp, PstI-ClaI-XhoI; 834 bp, 411 bp

EXAMPLE 21

[0141] Production of α-hANP Using E. coli H2:

[0142] α-hANP was obtained in a similar manner to those of Example 14and 15 using E. coli H2 in place of E. coli H1.

[0143] Amino acid sequence of thus obtained α-hANP was identical withthe known sequence of α-hANP.

1 88 1 28 PRT Homo sapiens 1 Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly ArgMet Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser Gly Leu Gly Cys Asn Ser PheArg Tyr 20 25 2 15 DNA Artificial Sequence synthetic DNA 2 ctgcgtagatcctct 15 3 18 DNA Artificial Sequence synthetic DNA 3 agcyygaagyygagcayg 18 4 18 DNA Artificial Sequence synthetic DNA 4 aattcatgctcaacttca 18 5 14 DNA Artificial Sequence synthetic DNA 5 aattcggtat gggc14 6 16 DNA Artificial Sequence synthetic DNA 6 ttcaccgccc ataccg 16 715 DNA Artificial Sequence synthetic DNA 7 ggtgaagcta aatct 15 8 14 DNAArtificial Sequence synthetic DNA 8 cgcagagatt tagc 14 9 15 DNAArtificial Sequence synthetic DNA 9 aagcaagagg atcta 15 10 15 DNAArtificial Sequence synthetic DNA 10 tgctttggtg gccgt 15 11 15 DNAArtificial Sequence synthetic DNA 11 tccatacggc cacca 15 12 15 DNAArtificial Sequence synthetic DNA 12 atggaccgca tcgct 15 13 15 DNAArtificial Sequence synthetic DNA 13 tgagcaccga tgcgg 15 14 15 DNAArtificial Sequence synthetic DNA 14 gctcagtccg gtctg 15 15 15 DNAArtificial Sequence synthetic DNA 15 cagcccagac cggac 15 16 15 DNAArtificial Sequence synthetic DNA 16 ggctgtaact ctttc 15 17 15 DNAArtificial Sequence synthetic DNA 17 taacggaaag agtta 15 18 12 DNAArtificial Sequence synthetic DNA 18 cgttactgat ag 12 19 11 DNAArtificial Sequence synthetic DNA 19 gatcctatca g 11 20 12 DNAArtificial Sequence synthetic DNA 20 aatttgccga ca 12 21 16 DNAArtificial Sequence synthetic DNA 21 cgttatgatg tcggca 16 22 16 DNAArtificial Sequence synthetic DNA 22 tcataacggt tctggc 16 23 16 DNAArtificial Sequence synthetic DNA 23 gaatatttgc cagaac 16 24 16 DNAArtificial Sequence synthetic DNA 24 aaatattctg aaatga 16 25 16 DNAArtificial Sequence synthetic DNA 25 tcaacagctc atttca 16 26 16 DNAArtificial Sequence synthetic DNA 26 gctgttgaca attaat 16 27 16 DNAArtificial Sequence synthetic DNA 27 gttcgatgat taattg 16 28 16 DNAArtificial Sequence synthetic DNA 28 catcgaacta gttaac 16 29 16 DNAArtificial Sequence synthetic DNA 29 gcgtactagt taacta 16 30 16 DNAArtificial Sequence synthetic DNA 30 tagtacgcaa gttcac 16 31 15 DNAArtificial Sequence synthetic DNA 31 cttttacgtg aactt 15 32 13 DNAArtificial Sequence synthetic DNA 32 gtaaaaaggg tat 13 33 7 DNAArtificial Sequence synthetic DNA 33 cgatacc 7 34 15 DNA ArtificialSequence synthetic DNA 34 gtaaaaaggg tatcg 15 35 11 DNA ArtificialSequence synthetic DNA 35 aattcgatac c 11 36 11 DNA Artificial Sequencesynthetic DNA 36 aattcatggc t 11 37 16 DNA Artificial Sequence syntheticDNA 37 ggttgtaaga acttct 16 38 13 DNA Artificial Sequence synthetic DNA38 tttggaagac ttt 13 39 16 DNA Artificial Sequence synthetic DNA 39cacttcgtgt tgatag 16 40 15 DNA Artificial Sequence synthetic DNA 40ttacaaccag ccatg 15 41 13 DNA Artificial Sequence synthetic DNA 41ccaaaagaag ttc 13 42 15 DNA Artificial Sequence synthetic DNA 42cgaagtgaaa gtctt 15 43 13 DNA Artificial Sequence synthetic DNA 43gatcctatca aca 13 44 11 DNA Artificial Sequence synthetic DNA 44aactagtacg c 11 45 16 DNA Artificial Sequence synthetic DNA 45aacttgcgta ctagtt 16 46 16 DNA Artificial Sequence synthetic DNA 46aagttcacgt aaaaag 16 47 16 DNA Artificial Sequence synthetic DNA 47ataccctttt tacgtg 16 48 15 DNA Artificial Sequence synthetic DNA 48ggtatcgata aaatg 15 49 16 DNA Artificial Sequence synthetic DNA 49gtagaacatt ttatcg 16 50 15 DNA Artificial Sequence synthetic DNA 50ttctacttca acaaa 15 51 15 DNA Artificial Sequence synthetic DNA 51ggtcggtttg ttgaa 15 52 13 DNA Artificial Sequence synthetic DNA 52ccgaccggct atg 13 53 15 DNA Artificial Sequence synthetic DNA 53gctggagcca tagcc 15 54 15 DNA Artificial Sequence synthetic DNA 54gctccagctc tcgtc 15 55 15 DNA Artificial Sequence synthetic DNA 55cggtgcgcga cgaga 15 56 15 DNA Artificial Sequence synthetic DNA 56gcgcaccgca gactg 15 57 12 DNA Artificial Sequence synthetic DNA 57gataccagtc tg 12 58 12 DNA Artificial Sequence synthetic DNA 58gtatcgtaga cg 12 59 12 DNA Artificial Sequence synthetic DNA 59accctcgtct ac 12 60 12 DNA Artificial Sequence synthetic DNA 60agggtggcga tg 12 61 11 DNA Artificial Sequence synthetic DNA 61aattcatcgc c 11 62 15 DNA Artificial Sequence synthetic DNA 62gatcctcgag atcaa 15 63 19 DNA Artificial Sequence synthetic DNA 63gcctttaatt gatctcgag 19 64 18 DNA Artificial Sequence synthetic DNA 64ttaaaggctc cttttgga 18 65 18 DNA Artificial Sequence synthetic DNA 65aaaaaggctc caaaagga 18 66 13 DNA Artificial Sequence synthetic DNA 66gccttttttt ttt 13 67 10 DNA Artificial Sequence synthetic DNA 67tcgacaaaaa 10 68 106 DNA Artificial Sequence synthetic trp promoter 68aattgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc 60atcgaactag ttaactagta cgcaagttca cgtaaaaagg gtatcg 106 69 164 DNAArtificial Sequence synthetic trp promoter 69 aatttgccga catcataacggttctggcaa atattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagtaacgcaagtt cacgtaaaaa gggtatcgaa ttcatggctg 120 gttgtaagaa cttcttttggaagactttca cttcgtgttg atag 164 70 105 DNA Artificial Sequence synthetictrp promoter 70 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgttgacaattaat 60 catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtat 105 71308 DNA Homo sapiens 71 atgttctact tcaacaaacc gaccggctat ggctccagctctcgtcgcgc accgcagact 60 ggtatcgtag acgagggtgg cgatgaattc atgtgttactgccaggaccc atatgtaaaa 120 gaagcagaaa accttaagaa atactttaat gcaggtcattcagatgtagc ggataatgga 180 actcttttct taggcatttt gaagaattgg aaagaggagagtgacagaaa aataatgcag 240 agccaaattg tctccttcta cttcaagctt gaagttgagcatgaattcgg tatgggcggt 300 gaagctaa 308 72 103 PRT Homo sapiens 72 MetPhe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Asp Lys Ile Met Gln 65 70 7580 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Gly His Gly Phe 85 9095 Gly Met Gly Gly Glu Ala Lys 100 73 134 DNA Homo sapiens 73 agcttgaagttgagcatgaa ttcggtatgg gcggtgaagc taaatctgtg cgtagatcct 60 cttgctttggtggccgtatg gaccgcatcg gtgctcagtc cggtctgggc tgtaactctt 120 tccgttactgatag 134 74 42 PRT Homo sapiens 74 Lys Leu Glu Val Glu His Glu Phe GlyMet Gly Gly Glu Ala Lys Ser 1 5 10 15 Leu Arg Arg Ser Ser Cys Phe GlyGly Arg Met Asp Arg Gly Ala Gln 20 25 30 Ser Gly Leu Gly Cys Asn Ser PheArg Tyr 35 40 75 4 PRT Homo sapiens 75 Ser Phe Arg Tyr 1 76 28 PRT Homosapiens 76 Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg IleGly 1 5 10 15 Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20 25 7784 DNA Homo sapiens 77 tctctgcgta gatcctcttg ctttggtggc cgtatggaccgcatcggtgc tcagtccggt 60 ctggggtgta actctttccg ttac 84 78 111 DNAArtificial Sequence synthetic DNA 78 aatttgccga catcataacg gttctggcaaatattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttcacgtaaaaag ggtatcgaag g 111 79 167 DNA Artificial Sequence synthetic DNA79 aatttgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat 60catcgaacta gttaactagt acgcaagttc acgtaaaaag ggtatcgaat tcatggctgg 120ttgtaagaac ttcttttgga agactttcac ttcgtgttga taggatc 167 80 107 DNAArtificial Sequence synthetic DNA 80 aatttgccga catcataacg gttctggcaaatattctgaa atgagctgtt gacaattaat 60 catcgaacta gttaactagt acgcaagttcacgtaaaaag ggtatcg 107 81 309 DNA Homo sapiens CDS (1)..(309) 81 atg ttctac ttc aac aaa ccg acc ggc tat ggc tcc agc tct cgt cgc 48 Met Phe TyrPhe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 gca ccgcag act ggt atc gta gac gag ggt ggc gat gaa ttc atg tgt 96 Ala Pro GlnThr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30 tac tgc caggac cca tat gta aaa gaa gca gaa aac ctt aag aaa tac 144 Tyr Cys Gln AspPro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45 ttt aat gca ggtcat tca gat gta gcg gat aat gga act ctt ttc tta 192 Phe Asn Ala Gly HisSer Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60 ggc att ttg aag aattgg aaa gag gag agt gac aga aaa ata atg cag 240 Gly Ile Leu Lys Asn TrpLys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 75 80 agc caa att gtc tccttc tac ttc aag ctt gaa gtt gag cat gaa ttc 288 Ser Gln Ile Val Ser PheTyr Phe Lys Leu Glu Val Glu His Glu Phe 85 90 95 ggt atg ggc ggt gaa gctaaa 309 Gly Met Gly Gly Glu Ala Lys 100 82 103 PRT Homo sapiens 82 MetPhe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 25 30Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 40 45Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 55 60Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 70 7580 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 85 9095 Gly Met Gly Gly Glu Ala Lys 100 83 138 DNA Homo sapiens CDS(3)..(131) 83 ag ctt gaa gtt gag cat gaa ttc ggt atg ggc ggt gaa gct aaatct 47 Leu Glu Val Glu His Glu Phe Gly Met Gly Gly Glu Ala Lys Ser 1 510 15 ctg cgt aga tcc tct tgc ttt ggt ggc cgt atg gac cgc atc ggt gct 95Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala 20 25 30cag tcc ggt ctg ggc tgt aac tct ttc cgt tac tga taggatc 138 Gln Ser GlyLeu Gly Cys Asn Ser Phe Arg Tyr 35 40 84 42 PRT Homo sapiens 84 Leu GluVal Glu His Glu Phe Gly Met Gly Gly Glu Ala Lys Ser Leu 1 5 10 15 ArgArg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln 20 25 30 SerGly Leu Gly Cys Asn Ser Phe Arg Tyr 35 40 85 128 DNA Artificial Sequencesynthetic DNA and Homo sapien hybrid 85 aactagtacg caagttcacg taaaaagggtatcgataaa atg ttc tac ttc aac 54 Met Phe Tyr Phe Asn 1 5 aaa ccg acc ggctat ggc tcc agc tct cgt cgc gca ccg cag act ggt 102 Lys Pro Thr Gly TyrGly Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly 10 15 20 atc gta gac gag ggtggc gat gaa gg 128 Ile Val Asp Glu Gly Gly Asp Glu 25 86 29 PRTArtificial Sequence synthetic DNA and Homo sapien hybrid 86 Met Phe TyrPhe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 10 15 Ala ProGln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu 20 25 87 393 DNA Homosapiens CDS (1)..(393) 87 atg ttc tac ttc aac aaa ccg acc ggc tat ggctcc agc tct cgt cgc 48 Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly SerSer Ser Arg Arg 1 5 10 15 gca ccg cag act ggt atc gta gac gag ggt ggcgat gaa ttc atg tgt 96 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly AspGlu Phe Met Cys 20 25 30 tac tgc cag gac cca tat gta aaa gaa gca gaa aacctt aag aaa tac 144 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn LeuLys Lys Tyr 35 40 45 ttt aat gca ggt cat tca gat gta gcg gat aat gga actctt ttc tta 192 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr LeuPhe Leu 50 55 60 ggc att ttg aag aat tgg aaa gag gag agt gac aga aaa ataatg cag 240 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile MetGln 65 70 75 80 agc caa att gtc tcc ttc tac ttc aag ctt gaa gtt gag catgaa ttc 288 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His GluPhe 85 90 95 ggt atg ggc ggt gaa gct aaa tct ctg cgt aga tcc tct tgc tttggt 336 Gly Met Gly Gly Glu Ala Lys Ser Leu Arg Arg Ser Ser Cys Phe Gly100 105 110 ggc cgt atg gac cgc atc ggt gct cag tcc ggt ctg ggc tgt aactct 384 Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser115 120 125 ttc cgt tac 393 Phe Arg Tyr 130 88 131 PRT Homo sapiens 88Met Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 1 5 1015 Ala Pro Gln Thr Gly Ile Val Asp Glu Gly Gly Asp Glu Phe Met Cys 20 2530 Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr 35 4045 Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu 50 5560 Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln 65 7075 80 Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Glu Val Glu His Glu Phe 8590 95 Gly Met Gly Gly Glu Ala Lys Ser Leu Arg Arg Ser Ser Cys Phe Gly100 105 110 Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys AsnSer 115 120 125 Phe Arg Tyr 130

We claim:
 1. A process for the production of α-hANP by (1) culturing amicroorganism transformed with an expression vector comprising asynthetic gene encoding an amino acid sequence of a protectivepeptide-fused α-hANP in a nutrient medium, (2) recovering the protectivepeptide-fused α-hANP from the cultured broth and (3) removing theprotective peptide portion of the protective peptide-fused α-hANP.
 2. Aprocess for the production of α-hANP of claim 1, in which themicroorganism is bacteria.
 3. A process for the production of α-hANP ofclaim 2, in which the bacteria is a strain belonging to the genusEscherichia.
 4. A process for the production of α-hANP of claim 3, inwhich the strain is Escherichia coli.
 5. A process for the production ofα-hANP of claim 1, in which α-hANP gene portion of the synthetic gene isrepresented by the following DNA sequence: Coding:    5′-TCT CTG CGT AGATCC TCT TGC TTT GGT Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCAGGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAGCCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3′ CCG ACA TTG AGAAAG GCA ATG-5′


6. A process for the production of α-hANP of claim 1, in which theprotective peptide-fused α-hANP has the amino acid sequence representedin FIG.
 17. 7. A process for the production of α-hANP of claim 1, theprotectice peptide portion of protective peptide-fused α-hANP is removedin the presence of API.
 8. A chemically synthsized gene encoding aminoacid sequence of α-hANP:H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH9. The chemically synthsized gene of claim 8, which is represented bythe following DNA sequence: Coding:    5′-TCT CTG CGT AGA TCC TCT TGCTTT GGT Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATGGAC CGC ATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTCAGG CCA GAC GGC TGT AAC TCT TTC CGT TAC-3′ CCG ACA TTG AGA AAG GCAATG-5′


10. A chemically synthesized protective peptide gene having DNA sequenceencoding lysine as C-terminal of the protective peptide.
 11. Thechemically synthesized protective peptide gene of claim 10, which isrepresented by the DNA sequence of FIG.
 4. 12. A recombinant vectorcomprising chemically synthesized α-hANP gene.
 13. A recombinant vectorof claim 13, in which the α-hANP gene is represented by the followingDNA sequence: Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGTNoncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGCATC GGT GCT CAG TCC GGT CTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCAGAC GGC TGT AAC TCT TTC CGT TAC-3′ CCG ACA TTG AGA AAG GCA ATG-5′


14. A recombinant vector comprising chemically synthesized generepresented by the DNA sequence of FIG.
 17. 15. A recombinant vectorcomprising chemically synthesized gene represented by the DNA sequenceof FIG.
 4. 16. A transformant comprising expression vector of chemicallysynthesized α-hANP gene.
 17. A transformant of claim 16, in which theα-hANP gene is presented by the following DNA sequence:Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGT Noncoding: 3′-AGA GACGCA TCT AGG AGA ACG AAA CCA GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGTCTG CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC GGC TGT AAC TCT TTCCGT TAC-3′ CCG ACA TTG AGA AAG GCA ATG-5′


18. A cleaving method of the fused-protein composed of a peptides havinga lysine between a protective peptide and a targent peptide in thepresence of API.
 19. Synthetic trp promoter III represented by the DNAsequence of FIG. 3.